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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-06-10 to 1991-06-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. However, there was no analysis of exposure concentrations.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Three test media were assessed: 1)by direct addition of the substance and also using stock solutions of the test substance prepared in 2)tertiary butyl alcohol (TBA) and 3)in dimethylsulfoxide (DMSO). The exposure period was extended to 96-hours (nominal)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Stock solutions of the test substance were prepared in tertiary butyl alcohol (TBA) and in dimethylsulfoxide (DMSO) and used to treat the test media prepared for independent tests (N.B. the highest concentrations in these tests was prepared by direct dispersion of the test substance into the algal growth medium). The final concentration of TBA and DMSO in each of the test substance concentrations was 100 μL/L. Test media were also prepared by direct addition followed by ultrasonication for 30 minutes.

- Controls: Dilution water, Dilution water + 100 μl/l TBA and Dilution water + 100 μl/l DMSO

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tertiary butyl alcohol (TBA) and Dimethylsulfoxide (DMSO)

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 μl/l
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM

- Strain: SAG 86.81

- Source (laboratory, culture collection): Collection of algal cultures, Institute for Plant Physiology, University of Göttingen, Germany.

- Method of cultivation: a pre-culture of algae in the exponential growth phase was prepared as described in the OECD test guideline using the medium described in Dutch Standard NPR 6505.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
nominal
Hardness:
not reported
Test temperature:
23 +/-1ºC
pH:
The pH of the test media in which algae were not present was in the range 8.0-8.3 throughout the test.

The pH of the test media that did contain algae was in the range 8.2-8.4 after 3 days and 8.4-9.4 after 4 days.
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations by direct addition: 0(Control), 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 101 mg/l.

Nominal concentrations using TBA solvent: 0(Control), 0(Control + 100 μl/l TBA), 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 126 mg/l. The latter concentration was prepared by direct dispersion of the test substance into the algal growth medium.

Nominal concentrations using DMSO solvent: 0(Control), 0(Control + 100 μl/l DMSO), 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 150 mg/l. The latter concentration was prepared by direct dispersion of the test substance into the algal growth medium.
Details on test conditions:
TEST SYSTEM

- Test vessel: Conical flask

- Type: covered with silicone sponge caps

- Material, size, headspace, fill volume: glass, 200 ml with 100 ml of test medium

- Aeration: none

- Renewal rate of test solution (frequency/flow rate): static

- Initial cells density: 10000 cells/mL

- Control end cell density: 440000 - 526000 cells/ml after approximately 72 hours, 1332000 - 2220000 cells/ml after approximately 96 hours

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 6

- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Algal growth medium prepare by adding nutrients and salts to Milli-Q filtered water. The medium was filter-sterilized before use.

- Culture medium different from test medium: no

- Intervals of water quality measurement: Start of the test and after 72 and 96 hours (nominal) incubation.


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 60-120 μmol/s/m2 from fluorescent lamps


EFFECT PARAMETERS MEASURED: cell numbers after 24, 48, 72 and 96 hours (nominal)

- Determination of cell concentrations: Coulter Counter TAII


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3.2

- Range finding study

- Test concentrations: 0.0003, 0.004, 0.06, 0.70 and 9.6 mg/l

- Results used to determine the conditions for the definitive study: Mean cell concentrations (cells/ml) after 72 hour incubation were: 1170000 (Control), 1350000 (Solvent control), 1360000 (0.0003 mg/l), 1020000 (0.004 mg/l), 700000 (0.06 mg/l), 700000 (0.7 mg/l) and 320000 (9.6 mg/l).
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
direct dispersion
Basis for effect:
growth rate
Remarks on result:
other: 99-270
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with TBA
Basis for effect:
growth rate
Remarks on result:
other: 150-210
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with DMSO
Basis for effect:
growth rate
Remarks on result:
other: 120-360
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
direct dispersion
Basis for effect:
biomass
Remarks on result:
other: 32-101
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with TBA
Basis for effect:
biomass
Remarks on result:
other: 32-126
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
98 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with DMSO
Basis for effect:
biomass
Remarks on result:
other: 32-150
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
direct dispersion
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with TBA
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with DMSO
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control: yes

- Observation of abnormalities: no abnormalities were observed
Reported statistics and error estimates:
ECx values with respect to growth rate were calculated using a parametric model developed by Kooijman et al. ECx values relating to growth rate were calculated using the method described in the OECD test guideline.

NOEC values were determined by visual comparison of the growth curves of Control and treated cultures.

Kooijman, S.A.L.M., A.O. Hanstveit and H. Oldersma. (1983). Parametric analysis of population growth in bioassays. Water Research, 17: 527-538.

The EC50 values determined for effects on growth rate are above the highest test concentrations.

Table 1. Test results - test media prepared by direct dispersion

 Nominal test substance concentration (mg/l)  Mean cell concentration after 92.5 hours incubation (cells/ml)
 0 (Control)  2075000
 0 (Control)  2225000
 0.032  2190000
 0.10  1901000
 0.32  1844000
 1.0  2008000
 3.2  2127000
 10  1982000
 32  1929000
 101  817000

Table 2. Test results - test media prepared using TBA as solvent

 Nominal test substance concentration (mg/l)  Mean cell concentration after 94 hours incubation (cells/ml)
 0 (Control)  1817000
 0 (TBA control)  1773000
 0.032  1735000
 0.10  1810000
 0.32  1913000
 1.0  1910000
 3.2  1931000
 10  2254000
 32  1999000
 126 (without TBA)  426000

Table 3. Test results - test media prepared using DMSO as solvent

 Nominal test substance concentration (mg/l)  Mean cell concentration after 95.5 hours incubation (cells/ml)
 0 (Control)  1922000
 0 (DMSO control)  2061000
 0.032  2212000
 0.10  2059000
 0.32  2055000
 1.0  2165000
 3.2 2006000 
 10  2062000
 32  1914000
 150 (without DMSO)  591000
Validity criteria fulfilled:
yes
Conclusions:
96-hour EC50 values in the range 160-200 mg/l have been determined for the effects of the test substance on growth rate of Desmodesmus subspicatus (tested as Scenedesmus subspicatus) cultures. These values have been determined by extrapolation to above the highest test concentrations. The method of test medium preparation did not have a significant effect on the test result. A NOEC of 32 mg/l has been estimated from the test results. The results are interpreted with reference to nominal concentrations of the test substance. However the test substance is susceptible to hydrolysis and it is likely that the test organisms were primarily exposed to a mixture of the parent compound and the hydrolysis products of the substance. The test results are consistent with those of a related study (TNO, 1992b).

Description of key information

Toxicity to algae: 96 hour ErC50 160 mg/l and 96 hour NOEC 32 mg/l (growth rate) (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:
160 mg/L
EC10 or NOEC for freshwater algae:
32 mg/L

Additional information

A 96 hour EC50 value of 160 mg/l and NOEC of 32 mg/l (nominal) have been determined for the effects of the registered substance on growth rate of Desmodesmus subspicatus (tested as Scenedesmus subspicatus).

The value was obtained by extrapolation to above the highest test concentration from results obtained in test media prepared by direct dispersion of the test substance. The use of other methods of test media preparation did not have a significant effect on the test result. A NOEC of 32 mg/l has been estimated from the test results.

In a separate study, conducted by the same laboratory, a 96 hour EC50 value of >10 mg/l and NOEC ≥10 mg/l have been determined for the effects of the test substance on growth rate and of Desmodesmus subspicatus.

In view of the test media preparation method and exposure regime it is likely that the test organisms were exposed to a mixture of the parent compound and the hydrolysis product.

Above a concentration of approximately 100 mg/l cross-linked condensation products could potentially form over time in the aqueous test media. No evidence of undissolved material (parent substance or precipitated products) has been reported in the study report for this test.

Refer to the discussion in the Section 6 endpoint summary (Section 7.0 of the CSR) for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.